Project Summary/Abstract Type 1 diabetes (T1D) is a T cell mediated autoimmune metabolic disease characterized by destruction of pancreatic beta cells. Prediabetic subjects can be identified by the presence of autoantibodies towards insulin, glutamate decarboxylase, tyrosine phosphatase-like protein IA-2 and zinc transporter 8 before disease onset. Subjects with two or more biochemically defined antibodies have an approximately 30% chance of progressing to clinical T1D within a 3 year period. However, it is still difficult to predict the subgroup within the ? 2 antibody positive group that will progress to develop disease. Preliminary data in our laboratory shows that there are clear differences in the frequency of recently activated autoreactive T cells in T1D and healthy control subjects. This proposal centers on assays that directly measure the frequency and surface phenotype of these recently activated islet antigen specific T cells, thereby providing a biomarker for predicting the subgroup that will have more immediate disease onset. We will pair these assays with the most up-to-date technology to study the TCR clonotypes and single cell transcriptomes of these autoreactive T cells at a single cell level. We will test the hypothesis that disease specific TCR clonotypes are expanded during disease development and these cells develop a disease associated signature as subjects progress to develop T1D. We will also address the question of whether there is a general dysregulation of regulatory T cells (Treg), which leads to the increase of the activation of islet antigen specific cell and effector T cells. Lastly, we will evaluate whether the presence of demethylated insulin DNA in serum, which is a putative biomarker for beta cell death, is correlated with increases in the frequency of activated autoreactive T cells in prediabetic subjects. All of these experiments will be carried out with samples from Trialnet Living BioBank and local clinics. Specific aims: Aim 1: Test the hypothesis that an increase in the frequency of recently activated autoreactive CD4+ and CD8+ T cells precedes the onset of clinical diabetes. Aim 2: Test the hypothesis that prediabetic subjects that progress to T1D acquire an expanded TCR repertoire of islet antigen specific T cells and that those T cells exhibit a distinct transcript signature. Aim 3: Test the hypothesis that progression toward T1D is accompanied by an imbalance between Treg function and effector T cell responsiveness and by periods of active beta cell destruction. Successful execution of this proposal will demonstrate that T cells can be used as a biomarker for monitoring disease risk and will illuminate important immunological pathways that are relevant in the progression from being autoantibody positive to development of overt diabetes. Our findings should also provide new insights for possible avenues of intervention to delay or halt disease progression.